Electrochemical Hydrogel Lithography of Calcium-Alginate Hydrogels

materials Communication

Electrochemical Hydrogel Lithography of Calcium-Alginate Hydrogels for Cell Culture Fumisato Ozawa 1,2 , Kosuke Ino 1, *, Hitoshi Shiku 1 and Tomokazu Matsue 1,2, * 1 2

*

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan; [email protected] (F.O.); [email protected] (H.S.) WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8579, Japan Correspondence: [email protected] (K.I.); [email protected] (T.M.); Tel.: +81-22-795-5872 (K.I.); +81-22-795-7209 (T.M.)

Academic Editors: Sandra Van Vlierberghe and Peter Dubruel Received: 31 July 2016; Accepted: 22 August 2016; Published: 31 August 2016

Abstract: Here we propose a novel electrochemical lithography methodology for fabricating calcium-alginate hydrogels having controlled shapes. We separated the chambers for Ca2+ production and gel formation with alginate with a semipermeable membrane. Ca2+ formed in the production chamber permeated through the membrane to fabricate a gel structure on the membrane in the gel formation chamber. When the calcium-alginate hydrogels were modified with collagen, HepG2 cells proliferated on the hydrogels. These results show that electrochemical hydrogel lithography is useful for cell culture. Keywords: electrodeposition; electrochemical hydrogel lithography; calcium-alginate hydrogel; cell culture

1. Introduction Several cell culture methods have recently been developed for tissue engineering. For example, a culture surface modified with a thermo-responsive polymer has been used to collect cells in the form of sheets by reducing the temperature from 37 ◦ C to 20 ◦ C, and the cell sheets have been used for tissue engineering [1]. Magnetic force has been used to accumulate magnetically-labeled cells on a non-adherent surface. The accumulated cells were collected as a three-dimensional (3D) tissue organ following removal of the magnetic force [2,3]. In an electrochemical approach, alkanethiol self-assembled monolayers (SAMs) modified with RGD peptides were used to collect cells as a sheet via reductive desorption of the SAMs [4,5]. All these methods have been used to fabricate 3D tissue organs for tissue engineering. Hydrogels have been used to provide scaffolds for tissue engineering. Calcium-alginate hydrogels are frequently used because they are formed by simply reacting alginate with Ca2+ in aqueous solution. Several methods have been developed for fabricating biocompatible scaffolds with special shapes from alginate hydrogels. For example, a microfluidic system has been used to mix a sodium alginate solution and a Ca2+ solution to fabricate spherical and linear calcium-alginate hydrogels [6]. In other reports, an alginate hydrogel without Ca2+ was fabricated by enzyme-induced oxidative coupling of alginates modified with phenyl groups [7]. An electrochemical method for the formation of calcium-alginate hydrogels has also been reported [8–13]. In this method, electrodes are inserted into a sodium alginate solution containing CaCO3 particles. H+ is generated near the electrode by the electrolysis of water, then the generated H+ reacts with the CaCO3 particles to release Ca2+ into the sodium alginate solution, resulting in deposition of calcium-alginate hydrogels on the electrode surface. In our previous study, tubular structures and microwell arrays of calcium-alginate hydrogel were constructed by electrodeposition [12,13]. However, mammalian cells on the electrodes were slightly Materials 2016, 9, 744; doi:10.3390/ma9090744

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damaged during electrochemical acidification [12,13]; in addition, carrying out electrodeposition only on the electrodes limits the applicability of the method to bioengineering. To solve these problems, we developed an indirect method called electrochemical hydrogel lithography for the electrodeposition of calcium-alginate hydrogels. Electrochemical methods have been previously used to pattern biomaterials on solid substrates to form bionic interfaces [14–16]. These methods use a microelectrode to electrochemically generate reactive chemicals that cause the local detachment of species from a substrate surface. Nishizawa and coworkers named this technique “biolithography”, and demonstrated two-dimensional cell attachment and proliferation on the surface treated by biolithography [14]. In contrast, the electrochemical hydrogel lithography method described here fabricates calcium-alginate hydrogels indirectly on an arbitrary area. The present method can provide 3D hydrogels appropriate for fabricating organs on chips, since 3D hydrogels can mimic in vivo environments. 2. Experimental Section We used a semipermeable membrane to separate the chamber for producing Ca2+ production chamber) by electrochemical acidification from the chamber for fabricating calcium-alginate hydrogels (gel formation chamber). This separation reduced cell damage caused by electrochemical acidification and allowed the hydrogels to be fabricated on arbitrary areas. The procedure for the electrochemical hydrogel lithography of calcium-alginate hydrogels is illustrated in Figure 1. Briefly, a 1% w/v sodium alginate solution was prepared by dissolving sodium alginate (Code No. 19-0995; Wako Pure Chemical Industries Ltd., Osaka, Japan) in a buffer solution containing 137 mM NaCl, 2.7 mM KCl, 8.5 mM Na2 HPO4 and 1.5 mM KH2 PO4 (PBS, pH 7.5, Wako Pure Chemical Industries Ltd., Osaka, Japan). HepG2 cells (1.0 × 106 cells/mL) were suspended in the alginate sodium solution, then the HepG2 cells were cultured according to our previous paper [13]. A 1% w/v CaCO3 -dispersed solution was prepared by dispersing CaCO3 in PBS. HepG2 cells (1.0 × 106 cells/mL) were suspended in the above sodium alginate solution. The 1% w/v CaCO3 -dispersed solution was placed in the Ca2+ production chamber, and the sodium alginate solution was added to the gel formation chamber. The two chambers were separated by a semipermeable cellulose membrane (UC24-32-100, Viskase Co. Inc., Lombard, IL, USA, MWCO: 14,000, pore size: 4–5 nm diameter, thickness: 30.5 µm) (Figure 1). The membrane prevents CaCO3 particles in the Ca2+ production chamber from passing through to the gel formation chamber, but Ca2+ is small enough to pass through the pores of the membrane. The membrane also prevents alginate in the gel formation chamber from passing through to the Ca2+ production chamber. Platinum (Pt) wire and plate electrodes were inserted into the Ca2+ production chamber and placed on the membrane, then a voltage of 3.1 V vs. or 4.0 V vs. the Pt plate was applied to the Pt wire electrode for 10–60 s to generate H+ by electrolysis of water. The CaCO3 particles reacted with the H+ near the Pt wire electrode to liberate Ca2+ . The generated Ca2+ diffused to the gel formation chamber thorough the membrane and cross-linked the alginate, resulting in formation of calcium-alginate hydrogels on the membrane (Figure 1). Thus, calcium-alginate hydrogels are fabricated by indirect electrodeposition. After hydrogel formation, the membrane was washed with PBS, turned upside down, and incubated in culture medium. (Ca2+

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Figure Schemes for for the hydrogel lithography of calcium-alginate hydrogels.hydrogels. The Figure 1. 1.Schemes theelectrochemical electrochemical hydrogel lithography of calcium-alginate CaCO3-dispersed solution and the sodium alginate solution were introduced above (Ca2+ production The CaCO3 -dispersed solution and the sodium alginate solution were introduced above chamber) and below (gel formation chamber) the membrane, respectively. Electrolysis of water at the (Ca2+ production chamber) and below (gel formation chamber) the membrane, respectively. Electrolysis Pt wire electrode produced Ca2+ near the electrode. The Ca2+ diffused to the sodium alginate solution of water at the Pt wire electrode produced Ca2+ near the electrode. The Ca2+ diffused to the through the membrane to form hydrogels. During electrochemical hydrogel lithography, the sodium alginate the membrane to form electrode was solution placed onthrough the membrane (A) or scanned (B). hydrogels. During electrochemical hydrogel lithography, the electrode was placed on the membrane (A) or scanned (B).

3. Results and Discussion

3. Results Discussion To and fabricate a line-shaped calcium-alginate hydrogel, a Pt wire electrode 200 μm in diameter was placed onathe membrane calcium-alginate (Figure 1A), then 3.1 V were applied for 60 s. The schematic To fabricate line-shaped hydrogel, a Pt wire electrode 200 µm illustration in diameter was and picture are shown in Figure S1. Figure 2A shows that a line-shaped hydrogel was fabricated on placed on the membrane (Figure 1A), then 3.1 V were applied for 60 s. The schematic illustration and the membrane. No hydrogel formed around the electrode because the electrode was inserted into the picture2+are shown in Figure S1. Figure 2A shows that a line-shaped hydrogel was fabricated on the Ca production chamber, which did not contain alginate. These results show that Ca2+ generated membrane. No hydrogel formed around the electrode because the electrode was inserted into the Ca2+ above the membrane diffused to the gel-forming chamber to form line-shaped hydrogels. Thus, 2+ generated above the production chamber, which did not contain alginate. Thesemethod resultsisshow that Ca electrochemical hydrogel lithography based on the above applicable to the fabrication of membrane diffused hydrogels to the gel-forming to membrane. form line-shaped hydrogels. electrochemical calcium-alginate indirectly chamber through the Figure 2B shows thatThus, the line-shaped hydrogel lithography based on the above method is applicable to the fabrication of calcium-alginate hydrogels can be overlapped to generate complex patterns, indicating that a complicated pattern can be fabricated by repeated deposition. We alsoFigure demonstrated thatthat HepG2 can be trapped insidecan be hydrogels indirectly through the membrane. 2B shows thecells line-shaped hydrogels the deposited hydrogel (Figure 2C). overlapped to generate complex patterns, indicating that a complicated pattern can be fabricated by hydrogel patterns were fabricated electrochemical hydrogel lithography using a repeatedControlled deposition. We also demonstrated that by HepG2 cells can be trapped inside the deposited scanning Pt wire electrode (Figure 1B). Figure 2D shows a Z-shaped hydrogel fabricated by scanning hydrogel (Figure 2C). a 500-μm-diameter Pt electrode on the membrane at approximately 10 mm/min. The present method Controlled hydrogel patterns were fabricated by electrochemical hydrogel lithography using has several advantages over previous electrochemical patterning methods, including biolithography. a scanning Pt wire electrode (Figure 1B). Figure 2D shows a Z-shaped hydrogel fabricated by scanning For example, the present method can be used to fabricate 3D tissue organs in hydrogels. Furthermore, a 500-µm-diameter Ptsuch electrode onand the growth membrane at approximately Thetopresent method various molecules, as drugs factors, can be embedded10inmm/min. the hydrogels allow the has several advantages over previous electrochemical patterning methods, including biolithography. fabrication of organs on chips. In addition, since the calcium-alginate hydrogel can be decomposed For example, the treatment, present method to fabricate 3D tissue in hydrogels. Furthermore, by a suitable the cellscan canbebeused collected after forming the organs tissue organs on the chip. We previously reported anas electrochemical patterning method in whichin hydrogel formation various molecules, such drugs and growth factors, can [12,13] be embedded the hydrogels to was allow the

fabrication of organs on chips. In addition, since the calcium-alginate hydrogel can be decomposed by a suitable treatment, the cells can be collected after forming the tissue organs on the chip. We previously reported an electrochemical patterning method [12,13] in which hydrogel formation was limited to the

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electrode surface, thus was unsuitable forwas fabricating flexible 3D cell-hydrogel patterns. In contrast, limited to the and electrode surface, and thus unsuitable for fabricating flexible 3D cell-hydrogel Materials 2016, 9, 744 4 of 7 the present electrochemical hydrogel lithography method is a flexible tool because hydrogels patterns. In contrast, the present electrochemical hydrogel lithography method is a flexible toolcan be fabricated areas by scanning an was electrode. because hydrogels can besurface, fabricated onthus arbitrary areas by scanning an electrode. limitedon toarbitrary the electrode and unsuitable for fabricating flexible 3D cell-hydrogel patterns. In contrast, the present electrochemical hydrogel lithography method is a flexible tool because hydrogels can be fabricated on arbitrary areas by scanning an electrode.

Figure 2. Images of calcium-alginate hydrogels obtained placing electrode and applying a Figure 2. Images of calcium-alginate hydrogels obtained by by placing an an electrode and applying a voltage voltage (A–C) or scanning an electrode (D). Image of a line-shaped hydrogel (A) and an overlapped (A–C) or scanning an electrode (D). Image of a line-shaped hydrogel (A) and an overlapped line-shaped line-shaped hydrogel (C) Microscope image of HepG2 cells inside thean hydrogel; (D)and Theapplying Z-shapeda Figure 2. (C) Images of (B); calcium-alginate hydrogels obtained electrode hydrogel (B); Microscope image of HepG2 cells inside by theplacing hydrogel; (D) The Z-shaped hydrogel hydrogel was fabricated by an scanning the(D). electrode. voltage (A–C) or scanning electrode Image of a line-shaped hydrogel (A) and an overlapped was fabricated by scanning the electrode. line-shaped hydrogel (B); (C) Microscope image of HepG2 cells inside the hydrogel; (D) The Z-shaped

After trapping HepG2 by cells insidethe theelectrode. deposited hydrogel, the calcium-alginate hydrogel was hydrogel was fabricated scanning 2+ from the gel using 2% EDTA in PBS, and then the cells were harvested dissolved by removing Cacells After trapping HepG2 inside the deposited hydrogel, the calcium-alginate hydrogel was to evaluate their viability trypan blue assay. Figure shows improved cell viability dissolved by removing Ca2+using from the gelthe using 2% EDTA in3PBS, then the cells wereusing harvested After trapping HepG2 cellsthe inside deposited hydrogel, theand calcium-alginate hydrogel was electrochemical hydrogel lithography (indirect electrodeposition) compared with the previous 2+ dissolved by removing fromthe thetrypan gel using 2% EDTA PBS, and the improved cells were harvested to evaluate their viabilityCa using blue assay. inFigure 3 then shows cell viability (direct electrodeposition) method (Figure This improved improvement is due tousing the toelectrochemical evaluate their viability using the trypan blue assay. FigureS2). 3 shows cell viability usingelectrodeposition hydrogel lithography (indirect electrodeposition) compared with the previous 2+-forming chamber) from the gel-forming chamber separation of electrochemical acidification (Ca electrochemical(direct hydrogel lithography (indirect electrodeposition) compared with the is previous electrodeposition electrodeposition) method (Figure S2). This improvement due to the and the longer diffusion of H+. However, the separation the efficiencyisfordue hydrogel electrodeposition (directlength electrodeposition) method (Figure will S2). reduce This improvement to the 2+ separation of electrochemical acidification (Ca -forming chamber) from the gel-forming chamber and formation. viability using electrochemical hydrogel lithography was slightly lower compared to 2+-forming separationCell of electrochemical acidification (Ca chamber) from the gel-forming chamber + the longer diffusion length(control), of H . However, thethe separation will reduce the efficiency for hydrogel conventional cell culture indicating that present electrochemical still +. However, and the longer diffusion length of H the separation will reduce thedeposition efficiency method for hydrogel formation. Cell viability using electrochemical hydrogel lithography was slightly lower compared to adversely cell culture, though the hydrogels with cells were deposited. formation.affects Cell viability usingeven electrochemical hydrogel lithography wasindirectly slightly lower compared to

conventional cell cell culture (control), indicating the present presentelectrochemical electrochemical deposition method conventional culture (control), indicatingthat that the deposition method still still adversely affects cellcell culture, even though withcells cellswere were indirectly deposited. adversely affects culture, even thoughthe thehydrogels hydrogels with indirectly deposited.

Figure 3. Cell viability using a conventional culture method (control), the previous electrodeposition method (Figure S2), and electrochemical hydrogel lithography (indirect electrodeposition). Figure 3. Cell viability using a conventional culture method (control), the previous electrodeposition

Figure 3. Cell viability using a conventional culture method (control), the previous electrodeposition method (Figure S2), and electrochemical hydrogel lithography (indirect electrodeposition). method (Figure S2), and electrochemical hydrogel lithography (indirect electrodeposition).


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MaterialsHepG2 2016, 9, 744cells did not adhere to the calcium-alginate hydrogels or proliferate 5 ofin 7 the Since gels, hydrogels were modified with collagen. To modify the hydrogel, 2 mg/mL collagen type I Since HepG2 cells did not adhere to the calcium-alginate hydrogels or proliferate in the gels, (Nitta Gelatin, Osaka, Japan) was added to the sodium alginate solution, and then electrochemical hydrogels were modified with collagen. To modify the hydrogel, 2 mg/mL collagen type I (Nitta hydrogel lithography was performed to fabricate calcium-alginate/collagen hydrogels (4.0 V for 10 s). Gelatin, Osaka, Japan) was added to the sodium alginate solution, and then electrochemical hydrogel A dotlithography array of calcium-alginate/collagen hydrogels was fabricated on the(4.0 membrane byAchanging was performed to fabricate calcium-alginate/collagen hydrogels V for 10 s). dot the position the 500-µm-diameterhydrogels Pt wire electrode (Figure The size of hydrogel array of of calcium-alginate/collagen was fabricated on 4A). the membrane by the changing the dot 6 cells/mL) was controlled by changing the voltage application time (Figure 4B). HepG2 cells (1 × 10 position of the 500-μm-diameter Pt wire electrode (Figure 4A). The size of the hydrogel dot was were controlled suspended the collagen/sodium alginate solution, andHepG2 then acells dot (1 pattern with HepG2 byin changing the voltage application time (Figure 4B). × 106 cells/mL) were was suspended in the collagen/sodium alginate solution,Although and then the a dot patterncells withwere HepG2 was fabricated by electrochemical hydrogel lithography. HepG2 successfully fabricated bycalcium-alginate/collagen electrochemical hydrogel lithography. thethe HepG2 cellscells were successfully embedded in the hydrogels Although (Figure 4C), HepG2 did not proliferate embedded in the calcium-alginate/collagen 4C), the HepG2 cells did noton proliferate within the hydrogels. Therefore, HepG2 hydrogels cells (1 ×(Figure 106 cells/mL) were seeded a dot array within the hydrogels. Therefore, HepG2 cells (1 × 106 cells/mL) were seeded on a dot array pattern of pattern of calcium-alginate/collagen hydrogels, incubated for 6 h, and then washed to remove calcium-alginate/collagen hydrogels, incubated for 6 h, and then washed to remove unattached cells unattached cells from the hydrogels. The HepG2 cells proliferated on the hydrogels after seven from the hydrogels. The HepG2 cells proliferated on the hydrogels after seven days of incubation days (Figure of incubation (Figure 4D,E). Thesethat results suggest that calcium-alginate/collagen 4D,E). These results suggest calcium-alginate/collagen hydrogels can be usedhydrogels for tissue can be used for tissue engineering. engineering.

Figure 4. Fabrication of calcium-alginate/collagen hydrogels. (A) Image of a 3 × 3 dot array of the

Figure 4. Fabrication of calcium-alginate/collagen hydrogels. (A) Image of a 3 × 3 dot array of the hydrogel; (B) Dependence of dot diameter of the hydrogels on voltage application time. HepG2 cells hydrogel; (B) Dependence of dot diameter of the hydrogels on voltage application time. HepG2 cells were embedded within the hydrogels (C); or seeded on the hydrogels and cultured for zero (D) and were embedded within the hydrogels (C); or seeded on the hydrogels and cultured for zero (D) and seven days (E). seven days (E).

Hydrogel microstructures have conventionally been constructed by optical methods [17,18]. The present method, based on electrochemical lithography, hasconstructed several advantages over conventional Hydrogel microstructures have conventionally been by optical methods [17,18]. optical methods. For example, perhaps unlike optical methods, electrochemical hydrogel lithography The present method, based on electrochemical lithography, has several advantages over conventional can use a turbid suspension of microparticles, such as magnetite nanoparticles. Furthermore, optical methods. For example, perhaps unlike optical methods, electrochemical hydrogel lithography electrochemical hydrogel lithography can be applied in a space surrounded by light-shielding can use a turbid suspension of microparticles, such as magnetite nanoparticles. Furthermore, materials, such as in vivo. In addition, electrochemical methods can be used to analyze cells electrochemical hydrogel lithography can bereported applied ainlarge-scale a space surrounded by light-shielding materials, embedded in 3D hydrogels. We previously integration (LSI)-based amperometric such sensor as in vivo. In addition, electrochemical methods can be used to analyze cells embedded consisting of 400 sensor elements for high-throughput cell analysis and bioimaging [19,20].in 3D

hydrogels. We previously reported a large-scale integration (LSI)-based amperometric sensor consisting of 400 sensor elements for high-throughput cell analysis and bioimaging [19,20]. The electrode-array


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system can be easily combined with electrochemical hydrogel lithography, allowing organ construction and cell analysis on the same electrochemical chip. Hydrogels are widely utilized in electrochemistry. Kang et al. reported a hydrogel pen using an electrochemical reaction for 3D printing [21]. By using the methodology, 3D metallic structures were successfully fabricated on the nanometer scale. In contrast, the present method can allow us to fabricate desired hydrogels. In the future, an electrochemical method will be utilized for 3D hydrogel printing. 4. Conclusions We have developed an electrochemical hydrogel lithography methodology for the indirect deposition of calcium-alginate hydrogels on a semipermeable membrane. The structure and shape of the hydrogels are controllable by first placing the electrode on the membrane, and scanning the electrode. Cells incorporated in the hydrogel structure showed improved viability compared to our previous reports. Modification of the calcium-alginate hydrogel with collagen enabled proliferation of HepG2 cells on the hydrogels. These results suggest that electrochemical hydrogel lithography of calcium-alginate hydrogels is useful for culturing cells. Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/9/9/744/s1. Figure S1: Schematic illustration and picture for electrochemical lithography. The electrode was manually positioned and scanned. For the precise control, an xyz-stage is necessary, Figure S2: Procedure for the direct electrodeposition of calcium-alginate hydrogels. Ca2+ is produced from the reaction between CaCO3 and electrolytically-generated H+ from water. The generated Ca2+ reacts with alginate directly, forming a hydrogel on the electrode. This method has been previously reported. Acknowledgments: This work was supported in part by Grants-in-Aid for Scientific Research (A) (Nos. 25248032 and 16H02280) and for Young Scientists (B) (No. 23760745) from the Japan Society for the Promotion of Science (JSPS). Author Contributions: F.O. and K.I. conceived and designed the experiments; F.O. performed the experiments; all authors analyzed the data; all authors wrote the paper. Conflicts of Interest: The authors declare that they have no conflict of interest.

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